PROJECT SUMMARY Cryptosporidium (Crypto) causes diarrheal disease worldwide, which can be life-threatening in immunocompromised hosts such as untreated HIV/AIDS patients and malnourished children in resource- constrained countries. However, there is no consistently effective treatment for cryptosporidiosis in these vulnerable populations. Our long-term goal is to understand the role of Crypto proteins involved in mediating host cell attachment and invasion in order to develop novel interventions for cryptosporidiosis. We have identified and characterized Clec, a unique Crypto protein with three characteristics of proteins involved in attachment and invasion. Clec is 1) a glycosaminoglycan (GAG)-binding protein with 2) mucin-like and 3) C- type lectin domains. The objective of this application is to elucidate the role of Clec in Crypto infection. Clec is an extracellular type 1 membrane protein, which is conserved in C. parvum, C. hominis and C. muris, is expressed during infection in vitro, localizes to the apical surface and dense granules of invasive stages, binds to cell surface GAGs, specifically heparan sulfate proteoglycans (HSPGs) on intestinal epithelial cells and blocks C. parvum attachment to and invasion of these cells via interactions with these GAGs. These findings, strongly suggest that Clec plays a role in Crypto infection by mediating attachment and invasion and raise the possibility that it may serve as a target for intervention. However, until recently, it was not possible to determine the role of Crypto proteins using molecular genetics. Our central hypothesis is that Clec mediates Crypto attachment to and invasion of intestinal epithelial cells in vitro and promotes infection in vivo by binding to cell surface heparan sulfate proteoglycans. In this application, we propose to test this hypothesis in vitro and in vivo using CRISPR/Cas9 mediated gene-targeting approaches recently pioneered by our collaborator, Dr. Boris Striepen. The Specific Aims are 1) to determine the role of C. parvum Clec (CpClec) in attachment to and invasion of human intestinal epithelial cells in vitro using gene-targeting approaches and 2) To determine the role of CpClec in infection in vivo using animal models of cryptosporidiosis. At the completion of these aims we expect to have established the role of CpClec in attachment and invasion in vitro and in infection in vivo using molecular genetics and complementary approaches. Future studies will be directed at determining the role of specific domains of Clec, and identifying and elucidating the roles of other GAG-binding Crypto proteins using gene-targeting approaches. The long-term goal is to determine whether CpClec and/or other GAG-binding proteins can serve as targets for drug or vaccine development.